Otitis media is a major health problem causing substantial morbidity and resulting in substantial health care expenditures. The causes and treatment of this disorder remain incompletely understood. Eustachian tube obstruction, viral and subsequent bacterial infection and immunity have each been suggested to contribute to the etiopathophysiology of this disorder. Recent work in our laboratories has indicated that concurrent mast cell activation and bacterial infection can synergistically interact to induce strong inflammatory changes in the middle ear. These preliminary data, as well as prior research in the middle ear and other systems, suggest that the mast cell may play an important role in the innate and cognate defense of the middle ear, and may also contribute to otitis media pathogenesis. In this proposal Drs. S. Wasserman, A. Ryan and D. Broide propose a series of integrated experiments employing genetically modified mice to validate this hypothesis and to elucidate the mechanism(s) by which this synergistic interaction occurs. Aim 1 of this proposal will define the synergistic interaction utilizing mast cell deficient mice, with and without, reconstitution of their middle ear mast cells. Aim 2 will examine the mechanisms by which bacterial products enhance mast cell mediated synergistic inflammation by exploring the Toll-like receptor (TLR) pathways of mast cells. In these experiments TLR 2, 4, and 9 deficient mast cells will be used to re-constitute middle ear mast cell populations of mast cell deficient mice and the effect of bacterial/ mast cell interactions defined. MyD88 deficient mice, defective in all TLR signaling will be used to define potential redundancies in these responses. Aim 3 will re-constitute middle ear mast cells with mast cell populations obtained from mice which are deficient in one or more mast cell mediators including histamine, leukotriene and tumor necrosis factor alpha to define the mediator(s) responsible for mast cell enhancement of inflammation induced in the presence of bacteria. Aim 4 will elucidate the leukocyte/endothelial mechanisms by which mast cell] bacterial interactions enhance inflammation, by direct observations of leukocyte behavior in genetically modified animals. Together these studies will expand our understanding of the role of mast cells in otitis media and may identify new targets for therapeutic intervention. [unreadable] [unreadable]